Analysis of molecular subtypes and antibiotic resistance in Treponema pallidum isolates from blood donors in Khyber Pakhtunkhwa, Pakistan

Syphilis, caused by Treponema pallidum, is resurging globally. Molecular typing allows for the investigation of its epidemiology. In Pakistan and other nations, T. pallidum subsp. pallidum has developed widespread macrolide resistance in the past decade. A study at the Peshawar Regional Blood Centre from June 2020–June 2021 analyzed serum samples from 32,812 blood donors in Khyber Pakhtunkhwa, Pakistan, to assess circulating T. pallidum strains and antibiotic resistance. Blood samples were initially screened for T. pallidum antibodies using a chemiluminescent microparticle immunoassay (CMIA). CMIA-reactive samples underwent polymerase chain reaction (PCR) targeted the polA, tpp47, bmp, and tp0319 genes. PCR-positive samples were further analyzed for molecular subtyping using a CDC-developed procedure and tp0548 gene examination. All PCR-positive samples were analyzed for the presence of point mutations A2058G and A2059G in 23S rRNA, as well as the G1058C mutation in 16S rRNA. These mutations are known to impart antimicrobial resistance to macrolides and doxycycline, respectively. Out of 32,812 serum samples, 272 (0.83%) were CMIA-reactive, with 46 being PCR-positive. Nine T. pallidum subtypes were identified, predominantly 14d/f. The A2058G mutation in 23S rRNA was found in 78% of cases, while G1058C in 16S rRNA and A2059G in 23S rRNA were absent. The research found donor blood useful for assessing T. pallidum molecular subtypes and antibiotic resistance, especially when chancres are not present. The prevalent subtype was 14d/f (51.85%), and the high macrolide resistance of 36 (78%) indicates caution in using macrolides for syphilis treatment in Khyber Pakhtunkhwa, Pakistan.

In 1998, CDC researchers developed a molecular strain typing method to study pathogen diversity and disease epidemiology, specifically targeting T. pallidum, the syphilis-causing agent [38].The CDC typing method and sequence analysis of tp0548 gene segment are widely used for subtyping T. pallidum [9].Over the last decade, molecular subtyping of T. pallidum has emerged to enhance local monitoring and managing of syphilis epidemics [9,38].This method improves syphilis comprehension, aids in identifying reinfection and relapse, and distinguishes between strains linked to particular symptoms [9].Early syphilis chancres may selfheal unnoticed.Identifying the syphilis pathogen, T. pallidum, is impractical in clinical settings, such as dark field microscopy.Bacterial burden peaks in the secondary stage [39].Collecting samples from chancres is more challenging than obtaining blood samples.The process relies on the accessibility of suitable health services for individuals with sexually transmitted infections to ensure sample collection and diagnosis.Blood samples are preferred for PCR diagnosis, resistance monitoring, and molecular subtyping of syphilis.While benzathine penicillin G is the standard treatment, the primary concern with benzathine penicillin G treatment is the availability issue and the sensitivity of some individuals to beta-lactam antibiotics, rather than drug resistance but antibiotic resistance can cause treatment failures.Azithromycin and doxycycline are alternative medications for patients allergic to penicillin or those who reject injections [40].Macrolide resistance is widespread in China, the US, Canada, Australia, and Europe [10, 18-20, 23, 24, 27, 29], and it is connected to the 23S rRNA gene of T. pallidum's A2058G and A2059G mutations [41,42].In 2014 European guidelines, doxycycline was recommended as an alternative treatment for latent syphilis when penicillin was not possible [40].
Research is required to examine G1058C mutations in spirochetes, particularly in Pakistani regions such as Khyber Pakhtunkhwa, where data on tetracycline resistance is lacking.
The aim of this research was to examine blood samples obtained from 272 blood donors in Khyber Pakhtunkhwa, Pakistan, who were previously identified as reactive for syphilis during our analysis conducted between June 2020 and June 2021 [37].The goal was to investigate the molecular epidemiology of syphilis and identify point mutations associated with resistance to doxycycline and macrolides.

Materials and methods
This research, conducted on the samples we had previously analysed from June 2020 to June 2021 [37], involved a prospective, cross-sectional investigation at the Peshawar Regional Blood Centre.The Institutional Ethics Committees of the Regional Blood Centre in Peshawar, Khyber Pakhtunkhwa, Pakistan, and Kohat University of Science and Technology in Kohat, Pakistan, approved this study.This centre is responsible for collecting blood donations from individuals in the Peshawar region and distributing prepared blood components to hospitals throughout Peshawar, which serves a population of 4.7 million people.Additionally, due to its proximity to the Afghan border, the centre also provides blood components to a substantial number of Afghan refugees.Khyber Pakhtunkhwa (KP) is a province located in the northwestern part of Pakistan, comprising 37 districts with a total population of 40.8 million.Peshawar, as the provincial capital and a well-developed city with advanced healthcare services, attracts residents from other districts who frequently seek superior healthcare.The blood donors included in this study were from various regions within KP, with a significant representation of Afghan ethnicities.

Clinical specimens
In this study, 5 mL of blood was extracted from each blood donor using plain tubes, specifically red-top tubes.All blood samples were collected with the explicit, informed consent of the donors.The serum from these red-top tubes was used to screen blood donors for syphilis using the CMIA technique [43].A total of 32,812 samples were subjected to this testing method.The CMIA was employed to detect antibodies against T. pallidum in the serum of the blood donors.Subsequently, all serum samples that tested reactive for T. pallidum using CMIA underwent additional molecular analysis.

DNA extraction from CMIA reactive serum samples by spin protocol
According to the protocols recommended by the manufacturer within 24 hours of collection, the QIAamp DNA Blood Mini Kit (QIAGEN) was used for the extraction of DNA from all CMIA reactive samples for antibodies to T. pallidum.For the extraction of DNA, 200 μl of serum was used.The extracted DNA was preserved in Buffer AE and stored in a freezer at −20˚C before further analysis [9].

Detection of T. pallidum DNA by PCR
Before subtyping and analysis for the discovery of markers specific for antibiotic resistance, all T. pallidum-reactive samples identified by CMIA were submitted to PCR using primers specific for the polA, tpp47, bmp, and tp0319 genes of T. pallidum.To avoid contamination, distinct rooms were utilized for reagent preparation, DNA extraction, sample amplification, and subsequent PCR product analysis.In this study, the positive control included DNA derived from T. pallidum SS14 and Nichols strains, provided by the US-CDC, while distilled water was utilised as the negative control.PCR experiments were carried out in a volume of 50 μL, with 5 μL of DNA template obtained from the test subjects' clinical materials or the controls [18].A 2.0% agarose gel was used to identify the amplicons of the PCR.The gels were operated at 100 V of current for one hour with a 100-bp ladder.Following staining with ethidium bromide, bands were visible on a UV transilluminator [44].Samples that tested PCR-positive for any of the specified genes were considered to contain T. pallidum DNA, and these samples were subsequently used to assess the molecular subtype of the DNA and its resistance to antibiotics.The primers and their sequences used to identify T. pallidum DNA by PCR are listed in Table 1.

Molecular subtyping of T. pallidum
Molecular subtyping was conducted on all PCR-positive specimens, and the specific primers used for this procedure are listed in Table 1.The molecular categorization of T. pallidum was achieved by combining three different analyses, which included the arp gene, tpr EGJ genes, and tp0548 gene [9,19,45].

Determining the number of 60-base pair repeats in the arp gene.
A particular primer was used in PCR to count the number of 60-bp repeats in the arp gene.The results of the PCR were separated using agarose gel electrophoresis.The size of each PCR product was determined using the Quantity One software (version 4.6.7;Biorad), which was compared to a 1-kb DNA ladder (Invitrogen), and the number of 60-bp repeats in the arp gene was determined by comparing the size of the amplified PCR product to the PCR product amplified from the Nichols strain of T. pallidum, which carries 14 repeats [38].

Performing a restriction fragment length polymorphism (RFLP) analysis of the tpr subfamily II (tpr EGJ) genes.
To amplify the tpr genes, nested PCR was used.The previously mentioned amplification procedure in Section 2.3 and the two primer pairs were applied [44].The tpr EGJ genes were amplified using an outer primer pair.Internal primers were used in the second round of PCR, using the first round's amplicon as a template.The nested PCR product was purified using a QIAquick PCR Purification Kit from Qiagen, digested with a quick restriction endonuclease, and then electrophoresed on a 2.0% agarose gel to determine the digestion pattern through comparison with previously published data [38].

Examining the variable regions in the tp0548 gene, as previously documented.
For the examination of the tp0548 gene's variable region from positive samples, the previously published primer set was utilized to amplify the variable region of the tp0548 gene.Using a QIAquick PCR Purification Kit from Qiagen, PCR products were purified before being Table 1.Primers employed in the molecular examination of T. pallidum.

Types of Analysis
Target Gene Forward Primer Reverse Primer

Characterization of drug resistance genes in T. pallidum
2.5.1.Detection of the 23S rRNA A2058G and A2059G mutations.The point mutations A2058G and A2059G within the 23S rRNA gene of T. pallidum were identified by analyzing DNA extracted from PCR-positive samples.PCR, accompanied by purification using the QIAquick PCR purification kit provided by Qiagen and subsequent digestion using MboII and BsaI enzymes from New England Biolabs [41], was employed to identify mutations in rRNA genes associated with antimicrobial resistance.The primer sequences are detailed in Table 1 [15].
2.5.2.Detection of the 16S rRNA G1058C mutation.Three PCR primers were employed to detect the G1058C point mutation within the 16S rRNA gene of T. pallidum.Table 1 provides details about these primers.After purification and sequencing, the obtained sequence was compared to the standard 16S rDNA sequence of the T. pallidum Nichols strain using DNA Star software [15].

Statistical analysis
For statistical analysis, we utilized SPSS Inc.'s Statistical Package for the Social Sciences, version 24.0 (Chicago, IL).Associations between categorical variables were identified using either the Fisher exact test or the chi-square test.Findings with a P-value below 0.05 were considered statistically significant [15].

Regional analysis of molecular subtypes
When these different molecular subtypes were analyzed based on their occurrence in different districts of Khyber Pakhtunkhwa, molecular subtype 14d/f was identified in districts Charsada, Mardan, Bannu, Khyber, Swat, Kohat, and Peshawar in 1, 2, 1, 1, 2, 1, and 6 blood donors, respectively.Molecular subtype 14d/g was identified in districts Kohat and Peshawar in 1 and 2 blood donors, respectively.Molecular subtype 15d/g was identified in districts Mardan and Peshawar in one blood donor each.Molecular subtype 12d/f was identified only in 1 blood donor from district Swat.Molecular subtype 14b/f was identified in districts Kohat and Peshawar in one blood donor each.Molecular sub-type 12e/a was identified only in 1 blood donor from district Swabi.Molecular subtype 11d/f was identified in districts Swabi and Peshawar in one blood donor each.Molecular sub-type 14e/f was identified only in 1 blood donor from district Peshawar.Molecular subtype 6d/f was identified only in 1 blood donor from district Dir Lower (Table 6).

Analysis of point mutations linked to antibiotic resistance
These 46 samples, initially positive for PCR, underwent additional testing to detect A2058G and A2059G point mutations in 23S rRNA, along with G1058C point mutations in 16S rRNA.Among the samples, a significant presence of the A2058G point mutation in 16S rRNA was observed in 36 cases (78%, P<0.001), indicating resistance to macrolides.However, neither the A2059G mutations in 16S rRNA associated with macrolide resistance nor the G1058C mutations in 16S rRNA linked to doxycycline resistance were detected in any sample (Table 7).

Discussion
Syphilis is a transmissible infection [46].The prevalence of syphilis has increased in Pakistan and globally; it is a widespread sexually transmitted disease (STD) both in underdeveloped and advanced nations [47][48][49].To our knowledge, this is the first study in Pakistan conducted on the molecular subtyping and antibiotics resistance of T. pallidum.To our knowledge, globally, this is the first study on blood donors in which serum has been used exclusively on a large scale, regional molecular subtyping and detection of antimicrobial resistance were carried out to provide enhanced surveillance of syphilis.This is an effective way to assess circulating T. pallidum in blood donors because blood donors are healthy individuals and chancres are usually invisible; they might be overlooked, ignored, or possibly not present.Chancres samples are difficult to collect.This approach also provides an effective insight into the status of antimicrobial resistance in T. pallidum locally.Currently, it is possible but challenging to cultivate T. pallidum in vitro using a tissue culture system, and to understand this pathogen; modern molecular biology techniques provide a valuable platform.The reactive cases of syphilis (T.pallidum) in the present study were 272 (0.83%).The findings in our study were higher when compared with the results of different studies conducted in some developed countries, including the USA (0.16%) [50], Italy (0.031%) [51], Israel (0.047%) [52], and Saudi Arabia (0.044%) [53].When comparing our results with studies from various African countries, the prevalence of syphilis in blood donors was much higher than the results of the present study, for example, in Burkina Faso (1.5%) [54], Nigeria (3.1%) [55], and Angola (20.0%) [56].Our results were slightly higher when compared with a study conducted by the Safe Blood Transfusion Programme of Pakistan, in which the prevalence of syphilis in Pakistani blood donors was 0.72% [31].
In comparison to PCR, the study discovered poor levels of specificity for serological testing.The rationale is that only the specific subspecies causing venereal syphilis are detectable in our PCR-based diagnosis.Overall, our findings demonstrated the effectiveness of PCR in detecting T. pallidum subsp.pallidum, in contrast to the inability of various serological tests to distinguish between venereal syphilis and other treponematoses [70,71].The use of a more precise technique, such as PCR, in the detection of venereal syphilis, might aid in lowering the number of infections acquired through a blood transfusion due to the high prevalence of transfusion- transmitted infectious diseases.In addition, we found that false positives from these serological tests could result in inaccurate prevalence estimates.Due to the difficulty of interpreting the results, the diagnosis is frequently challenging (false negatives).In Khyber Pakhtunkhwa, Pakistan, the majority of blood recipients are infants or pregnant women; hence, incorrect findings and interpretations brought on by a test's flaw would not only harm the patient but might also result in problems or the development of congenital syphilis.Despite the drawbacks of using blood samples for molecular epidemiological research, such as the presence of PCR-inhibitory substances and low bacterial load, this approach can significantly increase the pool of potential participants in subtyping evaluations, including those with asymptomatic, persistent infection.For the subtyping of T. pallidum DNA from serum, we used the Enhanced Centres for Disease Control and Prevention (ECDC) typing method.
In this study, blood donors from the majority of the districts, including Peshawar, Kohat, Swat, Khyber, Bannu, Mardan, and Charsada, were found to have subtype 14d/f, which was the most common (in 7 districts out of 9).Our results are comparable to those of other studies where the 14d/f subtype was identified as the most common, such as studies conducted in the United States and China [9], Buenos Aires, Argentina [11], Hunan, China [15], China [16], Lima, Peru [12], Shanghai, China [17], Seattle, Washington [10], and Paris, France [14].
Due to their effectiveness, relative affordability, and ease of oral administration, macrolides and doxycycline antibiotics have been frequently used to treat syphilis in non-pregnant individuals who are penicillin-allergic [77].Earlier research revealed regional differences in macrolides resistance to T. pallidum, demonstrating a high incidence of resistance in several wealthy nations.The use of single-dose azithromycin became increasingly popular for treating syphilis when syphilis rates in the USA started to rise in 2000.For partner-delivered treatment of sexual encounters, this regimen proved especially effective.However, failures of azithromycin therapy were discovered in San Francisco in 2002 [78].
In the current study, the major cities of Khyber Pakhtunkhwa, Pakistan, including Peshawar, Kohat, and Mardan, were found to have the highest number of macrolide-resistant cases: 14, 6, and 5, respectively.In the current study, Peshawar, the largest city in Khyber Pakhtunkhwa, Pakistan, with a population comprised of individuals from all over KP, Afghanistan, and other areas of Pakistan, had the greatest number of macrolide-resistant cases.People from Afghanistan travel frequently to other nations as well as back to their own.Similarly, as previously mentioned, there appear to be geographical variations in the prevalence of macrolide-resistant T. pallidum strains, with the largest prevalence occurring in major towns with active tourist industries (Sydney, Shanghai, Dublin, London, San Francisco, and Seattle) [79].In Pakistan, no such type of study had been conducted before.Studies on the frequency of macrolide-resistant T. pallidum in Asia are scarce [30,45].Even though the disease is more common and antibiotics are more frequently used in medical procedures here (and occasionally misused) [80].

Conclusion
We demonstrated that subtype 14d/f was the most prevalent among circulating T. pallidum strains in blood donors in Khyber Pakhtunkhwa, Pakistan, during the study period.Additionally, we demonstrated that the A2058G mutation, linked to macrolide resistance, was present in all these strains.However, the G1058C mutation, associated with decreased susceptibility to doxycycline, was undetected.Consequently, macrolide medications should not be used to treat syphilis in Khyber Pakhtunkhwa, Pakistan.On the other hand, further research is needed to determine whether doxycycline, a secondary antibiotic suggested by European guidelines for syphilis treatment [40], is susceptible to resistance.To adapt treatment strategies quickly to changes in the molecular epidemiology of the illness, we recommend monitoring the molecular subtypes and antibiotic resistance of successive T. pallidum DNA samples from syphilis patients across Pakistan.